金黄色葡萄球菌中Hfq蛋白生物学功能的研究
摘要
金黄色葡萄球菌是可以引发从皮肤炎症、肺炎到休克综合症等一系列疾病的革兰氏阳性致病菌,其通过表达各种表面粘附蛋白和毒力因子如溶血素、蛋白酶等引发各种疾病。金葡菌的治疗目前临床上多采用联合使用抗生素的办法,但由于金葡菌抗药性问题不能解决,致使许多抗生素变的无能为力。
sRNA(small RNA)是真核和原核细胞中广泛存在的一类非编码RNA,真核细胞中的sRNA-microRNA与靶基因互补调节特定基因的表达。在细菌中也存在类似的非编码小RNA,这一种sRNA通过互补配对与靶mRNA的5’UTR相互作用,影响靶mRNA的稳定性和翻译。一个sRNA可以与多个靶基因配对,同时多个sRNA也可以作用于同一个靶基因,它们发挥作用依赖于RNA伴侣分子Hfq (a host factor for RNA phage Qβ)。已有的研究结果表明,sRNA在细菌生长代谢及毒力调控过程中发挥着重要的作用。目前有关sRNA的研究主要集中于大肠杆菌(Escherichia coli),新近的研究结果表明在革兰氏阳性菌金黄色葡萄球菌(Staphylococcus aureus)及单核细胞增多性李司特菌(Listeria monocytogenes)中也发现了新的sRNA,其中部分位于细菌基因组的病原岛或者为致病菌株特有,提示这些sRNA可能参与了致病菌毒素的表达调控。金葡菌RNAIII(一种sRNA)已经被证实作用于多个毒力相关基因参与调控金葡菌的致病性。
具有RNA伴侣分子活性的Hfq蛋白最早在大肠杆菌中发现,其主要的生物学功能是通过形成六聚体与RNA结合来影响RNA的稳定性或者是通过辅助非编码小RNA与mRNA结合来调节靶基因的表达。目前在大肠杆菌中,超过30%的非编码小RNA能够与Hfq蛋白结合。Hfq蛋白本身具有ATPase活性,能够加速转录及翻译过程。大肠杆菌hfq的无义突变能够影响一系列蛋白的表达,导致细菌表型发生较大的变化,研究表明Hfq蛋白还能够影响多种细菌外毒素表达。
在Hfq发挥其生物学功能的过程中有多种蛋白的参与,例如PNP(polynucleotide phosphorylase)、PAPI(polyA polymerase I)以及RNaseE等通过与Hfq蛋白的结合来调控靶基因的表达。核糖体蛋白S1与RNAP (RNA polymerase)之间的结合也需要Hfq蛋白辅助,Hfq蛋白在这些蛋白发挥调控功能的过程中发挥着广泛而重要的作用。
本研究选择了金葡菌Hfq蛋白为主要研究对象,通过寻找和鉴定与其相互作用的蛋白质及其调控的RNA分子,阐明了Hfq蛋白在金葡菌中的重要生物学功能。本论文的主要研究内容包括①金葡菌hfq突变株制备及相关检测;②Hfq蛋白聚体形式及其伴侣分子活性的研究;③与Hfq相互作用蛋白质的寻找;④Hfq调控的RNA分子的寻找及其调控功能的研究。本研究所获得的具体结果如下: 1、金葡菌hfq突变株制备及相关检测。通过基因重组的方法获得hfq的突变体菌株,突变体对动物的致病性明显降低。基因芯片和蛋白质组的结果表明Hfq蛋白可以影响金葡菌内多个编码基因的表达。同时我们还发现多个基因间区的转录水平发生改变。由于细菌中的sRNA大多由基因间区转录,这表明Hfq蛋白通过调控功能基因和sRNA在金葡菌内的水平,来发挥其生物学作用。
2、Hfq蛋白聚体形式及其伴侣分子活性的研究。我们克隆表达了金黄色葡萄球菌来源的Hfq蛋白,并检测了不同理化条件下重组蛋白的聚体稳定性。研究结果发现重组蛋白在体外可以形成二聚体、四聚体和六聚体形式,进一步的结果表明不同聚体形成的机制可能不同。同时我们利用氨基酸替代的方法研究发现56位和63位酪氨酸是Hfq蛋白形成稳定的六聚体形式的关键氨基酸。RNA结合实验结果表明突变体蛋白不能够与RNA结合,从而证实Hfq蛋白的聚体形式是发挥其RNA伴侣分子功能的关键。
3、与Hfq相互作用蛋白质的寻找及鉴定。我们在实验中发现,TRAP(target of RAP)蛋白与聚体形式Hfq蛋白特异结合。TRAP是金葡菌内与其致病性相关的蛋白分子,TRAP抗体对金葡菌外毒素的产生以及对动物的致病性有着明显的抑制作用,但是其具体作用方式还存在争议。我们的实验结果表明TRAP蛋白通过C末端与Hfq结合,并可以稳定Hfq蛋白聚体形式。trap和hfq突变菌株致死性明显降低,芯片及双向电泳结果也显示trap和hfq突变菌株中存在一些共同变化的基因和外分泌蛋白,这提示两种蛋白的相互作用可能会通过调控一些基因的表达影响金葡菌的致病性。
已有文献报道RNaseIII参与了RNAIII与spa mRNA所形成的复合体中mRNA的降解,我们在体外实验中发现,RNaseIII可以与Hfq结合,二者结合后RNaseIII不能降解RNA,提示Hfq与RNaseIII的相互作用可能影响RNA在金葡菌中的代谢。
4、Hfq调控的RNA分子的寻找及其调控功能的研究。首先我们利用免疫共沉淀的方法证实了Hfq以聚体形式与RNAIII结合,在此基础上我们建立了一种新的免疫共沉淀类似的方法-IPL(Immunoprecipitation-Like),利用该方法钓取了RNAIII作用的靶基因,结果显示spa和hla mRNA是RNAIII的靶分子,这与文献报道相一致,同时我们还发现多个与金葡菌毒力相关的基因也是RNAIII的靶分子。
ssrA又称为tmRNA或10saRNA,是细菌中高度保守的一种sRNA分子,其主要的生物学功能是通过行使tRNA和mRNA的双重功能,使合成停滞或者中断的蛋白质被迅速释放并降解。与hfq突变体相似,ssrA突变体的表面颜色明显加深,凝胶阻滞及免疫共沉淀实验也表明,在Hfq辅助下ssrA可以直接调节色素相关基因crtMNmRNA的表达,或通过调节sigB蛋白的表达间接影响金葡菌表面色素的调控,揭示了在金葡菌中ssrA不仅可以行使tmRNA功能,还可以在Hfq蛋白辅助下,作为一种sRNA来调控不同靶基因的表达,影响金葡菌表面色素的合成。
利用IPL的方法,我们寻找到一些新的与Hfq结合的sRNA分子,并通过RACE的方法找到两条新的全长sRNA,分别命名为IP1和IP3,在不同培养条件下这两条sRNA表达上调,提示这两种sRNA与金葡菌应激反应相关,同时我们发现两种sRNA可能分别作用于多个靶基因。
总之,本研究在阐明Hfq蛋白对金葡菌致病性的重要影响以及聚体形式对其RNA伴侣分子活性具有重要作用的基础上,寻找并鉴定了与其相关的蛋白和RNA分子,进一步研究了其在金葡菌中的分子生物学功能。Hfq蛋白在金葡菌中生物学功能的阐明有助于加深对金葡菌致病性的了解认识,为抗金葡菌感染提供更为深入的理论基础和新的药物靶标,也会为其它抗微生物感染的研究提供参考和借鉴。
The Gram-positive bacterium Staphylococcus aureus (S.aureus) is an important pathogen which can expressed many kinds of extrotoxins to cause a variety of diseases, such as cutaneous infection, pneumonia, toxic shock syndrome etc. Because most of S.aureus are drug-resistant, many antibiotics are useless.
sRNA (small RNA), a kind of non-coding RNA, exists in the eukaryote and prokaryocyte. The microRNA is a kind of sRNA in eukaryote and negatively regulates the target gene by annealing to mRNA. In bacteria, one kind of sRNA similar to microRNA, can regulate the expression of target gene by paring with the 5’UTR of mRNA. In bacteria, one sRNA can inteact with several targets and one gene can be regulated by multiple sRNAs. Hfq (a host factor for RNA phage Qβ), is necessary for sRNA interacting with its targets. Many reports showed that sRNAs involved in the process of metabolism and pathogenesis. Though the studies of sRNA mostly focus on the Escherichia coli (E.coli), some novel sRNAs are identified in S.aureus and Listeria monocytogenes. These sRNAs are predicted to regulate the pathogenesis because they are located in the pathogenicity islands or only expressed in the pathogenic strains. RNAIII is a small RNA, which is identified as a key regulator of toxin expression in S. aureus.
The Hfq was firstly discovered in E.coli. It can form a homohexameric ring to bind RNA and change the stability of RNA or help sRNA pairing with the target mRNA. Hfq also has ATPase activity. In E.coli, the expression of many proteins is altered in the hfq mutant comparing with wild type and the phenotype of mutant is different with wild type. It is reported that Hfq can influence the expression of exotoxin in some bacteria.
There are some proteins interacting with Hfq in E.coli, such as PNP(polynucleotide phosphorylase), PAPI(polyA polymerase I) and RNaseE. These proteins are important for Hfq.
In our study, we try to discover the biological function of Hfq in S.aureus from four parts. The first part is the preparation of hfq mutant. In the second part, we try to study the polymer formation and chaperone activity of recombinant Hfq protein. In the third part, the proteins interacting with Hfq are identified. We try to discover biological function of the sRNAs binding to Hfq in the last part. The main results of this study as following:
1. The preparation of hfq mutant. The mutant of hfq in S.aureus was obtained through gene recombination. It was found that the mutant was more harmless comparing with wild type in the animal model. The results of gene chip and proteomics showed that the level of many coding genes and intergenic sequences was changed in the mutant. Most sRNAs are transcribed from the intergenic region, so our results suggested that the Hfq protein could influence the expression of mRNA and sRNA in S.aureus.
2. The study of the polymer formation and chaperone activity of Hfq. The Hfq protein was recombinant expressed. The recombinant protein had the ATPase activity to accelerate the transcription of mRNA in vitro. The formation of Hfq polymer was tested in the different conditions. It was found that there were different kinds of forms of Hfq protein including monomer, dimmer, tetramer and hexamer. In the further study, we found that the formation mechanism of the different polymers was different. The tyrosine 56 and tyrosine 63 of Hfq protein were indentified as the the key amino acids for the polymer formation by using site mutation method. The result of the monomer of Hfq protein could not bind RNA suggested that the polymer structure was essential to its RNA chaperone function.
3. Identification of the interaction proteins of Hfq. First, we found that TRAP (target of RAP) could specifically bind the Hfq protein. TRAP is reported to be involved in the pathogenesis of S.aureus. The polyclonal antibody of TRAP can decrease the level of exotoxins of S.aures. In the further study, we found that TRAP could interact with Hfq though its C terminus and the TRAP protein could stable the polymer of Hfq. The pathogenesis of trap mutant is decreased. The results of gene chip and proteomics indicated that the expression of many genes and exoproteins was altered in the trap mutant. Many of them are also changed in the hfq mutant. These results suggested that the interaction of Hfq and TRAP could regulate some genes expression and influence the pathogenicity of S.aureus.
Previously study showed RNaseIII (endoribonuclease III) could rapidly degrades the spa mRNA in the complex of RNAIII/spa mRNA. In our experiment, we found that Hfq could specifically bind RNaseIII and block RNaseIII to degrade the RNA. It suggested that the interaction of Hfq and RNaseIII might involve in the metabolism of RNA in S. aureus.
4. Indentification of the function of the sRNAs which could bind to Hfq. We established a novel method named IPL(Immunoprecipitation-Like) to discover the Hfq binding sRNAs and their targets. Using this method, we found that the spa and hla mRNA were the targets of RNAIII, which was accordant to the previous reports. In the further study, we discovered some genes related to S.aureus pathogenesis as the novel targets of RNAIII.
ssrA (also called 10Sa and tmRNA) is highly conserved in many kinds of bacteria. In E. coli, ssrA bears properties of both an alanine-tRNA and an mRNA. By a mechanism called trans-translation, ssrA tags incomplete proteins expressed from broken or cleaved mRNA lacking in-frame stop codons The pigment of ssra mutant is up-regulated as same as the hfq mutant. Our results showed that ssrA could act as an antisense RNA to control the expression of CrtM/N and sigB with the aid of Hfq protin.These results indicated that ssrA could also act as a sRNA to regulate some genes expression.
We also found some novel sRNA candidates which could specifically bind to Hfq. Two full length novel sRNA were identified by using RACE which named as IP1 and IP3. IP1 and IP3 were up-regulated in the some stress condition. It suggested that these two sRNAs might be important for the stress response of S.aureus. Several genes were indenfied as the potential target of the two sRNAs.
In summary, our results showed that Hfq was an important protein in S.aureus. There were some proteins and sRNAs could interact with Hfq. Hfq protein was essential for the complex formation of sRNA and its targets. Many genes were regulated directly or indirectly by Hfq and its interacting molecules. Identification of Hfq function in S.aureus is important for the study of anti-infection in the future..
sRNA(small RNA)是真核和原核细胞中广泛存在的一类非编码RNA,真核细胞中的sRNA-microRNA与靶基因互补调节特定基因的表达。在细菌中也存在类似的非编码小RNA,这一种sRNA通过互补配对与靶mRNA的5’UTR相互作用,影响靶mRNA的稳定性和翻译。一个sRNA可以与多个靶基因配对,同时多个sRNA也可以作用于同一个靶基因,它们发挥作用依赖于RNA伴侣分子Hfq (a host factor for RNA phage Qβ)。已有的研究结果表明,sRNA在细菌生长代谢及毒力调控过程中发挥着重要的作用。目前有关sRNA的研究主要集中于大肠杆菌(Escherichia coli),新近的研究结果表明在革兰氏阳性菌金黄色葡萄球菌(Staphylococcus aureus)及单核细胞增多性李司特菌(Listeria monocytogenes)中也发现了新的sRNA,其中部分位于细菌基因组的病原岛或者为致病菌株特有,提示这些sRNA可能参与了致病菌毒素的表达调控。金葡菌RNAIII(一种sRNA)已经被证实作用于多个毒力相关基因参与调控金葡菌的致病性。
具有RNA伴侣分子活性的Hfq蛋白最早在大肠杆菌中发现,其主要的生物学功能是通过形成六聚体与RNA结合来影响RNA的稳定性或者是通过辅助非编码小RNA与mRNA结合来调节靶基因的表达。目前在大肠杆菌中,超过30%的非编码小RNA能够与Hfq蛋白结合。Hfq蛋白本身具有ATPase活性,能够加速转录及翻译过程。大肠杆菌hfq的无义突变能够影响一系列蛋白的表达,导致细菌表型发生较大的变化,研究表明Hfq蛋白还能够影响多种细菌外毒素表达。
在Hfq发挥其生物学功能的过程中有多种蛋白的参与,例如PNP(polynucleotide phosphorylase)、PAPI(polyA polymerase I)以及RNaseE等通过与Hfq蛋白的结合来调控靶基因的表达。核糖体蛋白S1与RNAP (RNA polymerase)之间的结合也需要Hfq蛋白辅助,Hfq蛋白在这些蛋白发挥调控功能的过程中发挥着广泛而重要的作用。
本研究选择了金葡菌Hfq蛋白为主要研究对象,通过寻找和鉴定与其相互作用的蛋白质及其调控的RNA分子,阐明了Hfq蛋白在金葡菌中的重要生物学功能。本论文的主要研究内容包括①金葡菌hfq突变株制备及相关检测;②Hfq蛋白聚体形式及其伴侣分子活性的研究;③与Hfq相互作用蛋白质的寻找;④Hfq调控的RNA分子的寻找及其调控功能的研究。本研究所获得的具体结果如下: 1、金葡菌hfq突变株制备及相关检测。通过基因重组的方法获得hfq的突变体菌株,突变体对动物的致病性明显降低。基因芯片和蛋白质组的结果表明Hfq蛋白可以影响金葡菌内多个编码基因的表达。同时我们还发现多个基因间区的转录水平发生改变。由于细菌中的sRNA大多由基因间区转录,这表明Hfq蛋白通过调控功能基因和sRNA在金葡菌内的水平,来发挥其生物学作用。
2、Hfq蛋白聚体形式及其伴侣分子活性的研究。我们克隆表达了金黄色葡萄球菌来源的Hfq蛋白,并检测了不同理化条件下重组蛋白的聚体稳定性。研究结果发现重组蛋白在体外可以形成二聚体、四聚体和六聚体形式,进一步的结果表明不同聚体形成的机制可能不同。同时我们利用氨基酸替代的方法研究发现56位和63位酪氨酸是Hfq蛋白形成稳定的六聚体形式的关键氨基酸。RNA结合实验结果表明突变体蛋白不能够与RNA结合,从而证实Hfq蛋白的聚体形式是发挥其RNA伴侣分子功能的关键。
3、与Hfq相互作用蛋白质的寻找及鉴定。我们在实验中发现,TRAP(target of RAP)蛋白与聚体形式Hfq蛋白特异结合。TRAP是金葡菌内与其致病性相关的蛋白分子,TRAP抗体对金葡菌外毒素的产生以及对动物的致病性有着明显的抑制作用,但是其具体作用方式还存在争议。我们的实验结果表明TRAP蛋白通过C末端与Hfq结合,并可以稳定Hfq蛋白聚体形式。trap和hfq突变菌株致死性明显降低,芯片及双向电泳结果也显示trap和hfq突变菌株中存在一些共同变化的基因和外分泌蛋白,这提示两种蛋白的相互作用可能会通过调控一些基因的表达影响金葡菌的致病性。
已有文献报道RNaseIII参与了RNAIII与spa mRNA所形成的复合体中mRNA的降解,我们在体外实验中发现,RNaseIII可以与Hfq结合,二者结合后RNaseIII不能降解RNA,提示Hfq与RNaseIII的相互作用可能影响RNA在金葡菌中的代谢。
4、Hfq调控的RNA分子的寻找及其调控功能的研究。首先我们利用免疫共沉淀的方法证实了Hfq以聚体形式与RNAIII结合,在此基础上我们建立了一种新的免疫共沉淀类似的方法-IPL(Immunoprecipitation-Like),利用该方法钓取了RNAIII作用的靶基因,结果显示spa和hla mRNA是RNAIII的靶分子,这与文献报道相一致,同时我们还发现多个与金葡菌毒力相关的基因也是RNAIII的靶分子。
ssrA又称为tmRNA或10saRNA,是细菌中高度保守的一种sRNA分子,其主要的生物学功能是通过行使tRNA和mRNA的双重功能,使合成停滞或者中断的蛋白质被迅速释放并降解。与hfq突变体相似,ssrA突变体的表面颜色明显加深,凝胶阻滞及免疫共沉淀实验也表明,在Hfq辅助下ssrA可以直接调节色素相关基因crtMNmRNA的表达,或通过调节sigB蛋白的表达间接影响金葡菌表面色素的调控,揭示了在金葡菌中ssrA不仅可以行使tmRNA功能,还可以在Hfq蛋白辅助下,作为一种sRNA来调控不同靶基因的表达,影响金葡菌表面色素的合成。
利用IPL的方法,我们寻找到一些新的与Hfq结合的sRNA分子,并通过RACE的方法找到两条新的全长sRNA,分别命名为IP1和IP3,在不同培养条件下这两条sRNA表达上调,提示这两种sRNA与金葡菌应激反应相关,同时我们发现两种sRNA可能分别作用于多个靶基因。
总之,本研究在阐明Hfq蛋白对金葡菌致病性的重要影响以及聚体形式对其RNA伴侣分子活性具有重要作用的基础上,寻找并鉴定了与其相关的蛋白和RNA分子,进一步研究了其在金葡菌中的分子生物学功能。Hfq蛋白在金葡菌中生物学功能的阐明有助于加深对金葡菌致病性的了解认识,为抗金葡菌感染提供更为深入的理论基础和新的药物靶标,也会为其它抗微生物感染的研究提供参考和借鉴。
The Gram-positive bacterium Staphylococcus aureus (S.aureus) is an important pathogen which can expressed many kinds of extrotoxins to cause a variety of diseases, such as cutaneous infection, pneumonia, toxic shock syndrome etc. Because most of S.aureus are drug-resistant, many antibiotics are useless.
sRNA (small RNA), a kind of non-coding RNA, exists in the eukaryote and prokaryocyte. The microRNA is a kind of sRNA in eukaryote and negatively regulates the target gene by annealing to mRNA. In bacteria, one kind of sRNA similar to microRNA, can regulate the expression of target gene by paring with the 5’UTR of mRNA. In bacteria, one sRNA can inteact with several targets and one gene can be regulated by multiple sRNAs. Hfq (a host factor for RNA phage Qβ), is necessary for sRNA interacting with its targets. Many reports showed that sRNAs involved in the process of metabolism and pathogenesis. Though the studies of sRNA mostly focus on the Escherichia coli (E.coli), some novel sRNAs are identified in S.aureus and Listeria monocytogenes. These sRNAs are predicted to regulate the pathogenesis because they are located in the pathogenicity islands or only expressed in the pathogenic strains. RNAIII is a small RNA, which is identified as a key regulator of toxin expression in S. aureus.
The Hfq was firstly discovered in E.coli. It can form a homohexameric ring to bind RNA and change the stability of RNA or help sRNA pairing with the target mRNA. Hfq also has ATPase activity. In E.coli, the expression of many proteins is altered in the hfq mutant comparing with wild type and the phenotype of mutant is different with wild type. It is reported that Hfq can influence the expression of exotoxin in some bacteria.
There are some proteins interacting with Hfq in E.coli, such as PNP(polynucleotide phosphorylase), PAPI(polyA polymerase I) and RNaseE. These proteins are important for Hfq.
In our study, we try to discover the biological function of Hfq in S.aureus from four parts. The first part is the preparation of hfq mutant. In the second part, we try to study the polymer formation and chaperone activity of recombinant Hfq protein. In the third part, the proteins interacting with Hfq are identified. We try to discover biological function of the sRNAs binding to Hfq in the last part. The main results of this study as following:
1. The preparation of hfq mutant. The mutant of hfq in S.aureus was obtained through gene recombination. It was found that the mutant was more harmless comparing with wild type in the animal model. The results of gene chip and proteomics showed that the level of many coding genes and intergenic sequences was changed in the mutant. Most sRNAs are transcribed from the intergenic region, so our results suggested that the Hfq protein could influence the expression of mRNA and sRNA in S.aureus.
2. The study of the polymer formation and chaperone activity of Hfq. The Hfq protein was recombinant expressed. The recombinant protein had the ATPase activity to accelerate the transcription of mRNA in vitro. The formation of Hfq polymer was tested in the different conditions. It was found that there were different kinds of forms of Hfq protein including monomer, dimmer, tetramer and hexamer. In the further study, we found that the formation mechanism of the different polymers was different. The tyrosine 56 and tyrosine 63 of Hfq protein were indentified as the the key amino acids for the polymer formation by using site mutation method. The result of the monomer of Hfq protein could not bind RNA suggested that the polymer structure was essential to its RNA chaperone function.
3. Identification of the interaction proteins of Hfq. First, we found that TRAP (target of RAP) could specifically bind the Hfq protein. TRAP is reported to be involved in the pathogenesis of S.aureus. The polyclonal antibody of TRAP can decrease the level of exotoxins of S.aures. In the further study, we found that TRAP could interact with Hfq though its C terminus and the TRAP protein could stable the polymer of Hfq. The pathogenesis of trap mutant is decreased. The results of gene chip and proteomics indicated that the expression of many genes and exoproteins was altered in the trap mutant. Many of them are also changed in the hfq mutant. These results suggested that the interaction of Hfq and TRAP could regulate some genes expression and influence the pathogenicity of S.aureus.
Previously study showed RNaseIII (endoribonuclease III) could rapidly degrades the spa mRNA in the complex of RNAIII/spa mRNA. In our experiment, we found that Hfq could specifically bind RNaseIII and block RNaseIII to degrade the RNA. It suggested that the interaction of Hfq and RNaseIII might involve in the metabolism of RNA in S. aureus.
4. Indentification of the function of the sRNAs which could bind to Hfq. We established a novel method named IPL(Immunoprecipitation-Like) to discover the Hfq binding sRNAs and their targets. Using this method, we found that the spa and hla mRNA were the targets of RNAIII, which was accordant to the previous reports. In the further study, we discovered some genes related to S.aureus pathogenesis as the novel targets of RNAIII.
ssrA (also called 10Sa and tmRNA) is highly conserved in many kinds of bacteria. In E. coli, ssrA bears properties of both an alanine-tRNA and an mRNA. By a mechanism called trans-translation, ssrA tags incomplete proteins expressed from broken or cleaved mRNA lacking in-frame stop codons The pigment of ssra mutant is up-regulated as same as the hfq mutant. Our results showed that ssrA could act as an antisense RNA to control the expression of CrtM/N and sigB with the aid of Hfq protin.These results indicated that ssrA could also act as a sRNA to regulate some genes expression.
We also found some novel sRNA candidates which could specifically bind to Hfq. Two full length novel sRNA were identified by using RACE which named as IP1 and IP3. IP1 and IP3 were up-regulated in the some stress condition. It suggested that these two sRNAs might be important for the stress response of S.aureus. Several genes were indenfied as the potential target of the two sRNAs.
In summary, our results showed that Hfq was an important protein in S.aureus. There were some proteins and sRNAs could interact with Hfq. Hfq protein was essential for the complex formation of sRNA and its targets. Many genes were regulated directly or indirectly by Hfq and its interacting molecules. Identification of Hfq function in S.aureus is important for the study of anti-infection in the future..
引文
[1] Lowy FD.Staphylococcus aureus infections. N Engl J Med,1998,339:520-532.
[2] Franze MT, Eoyang L, August JT. Factor fraction required for the synthesis of bacteriophage Qb-RNA.Nature,1968, 219(5154): 588-590.
[3] Moller T, Franch T, Hojrup P, et al.Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell , 2002, 9(1):23-30.
[4] Zhang A, Wassarman KM, Ortega J, et al.The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell ,2002,9(1):11-22.
[5] Sledjeski DD, Whitman C, Zhang A. Hfq is necessary for regulation by the untranslated RNA DsrA. J Bacteriol , 2001,183(6):1997-2005.
[6] Sun X, Zhulin I, Wartell R . Predicted structure and phyletic distribution of the RNA-binding protein Hfq. Nucleic Acids Res, 2002, 30(17):3662-3671.
[7] Schumacher MA, Pearson RF, Moller T, et al. Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J , 2002, 21(13):3546-3556.
[8] Mohanty BK, Maples VF, Kushner SR. The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Mol Microbiol, 2004, 54(4):905–920.
[9] Le Derout J, Folichon M, Briani F, et al.Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs. Nucleic Acids Res, 2003, 31(14):4017–4023.
[10] Hajnsdorf E, Re′gnier P. Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I. Proc Natl Acad Sci USA, 2000, 97(4):1501–1505.
[11] Vytvytska O, Jakobsen JS, Balcunate G, et al.Host factor I, Hfq binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability. Proc Natl Acad Sci USA, 1998, 95(24):14118–14123.
[12] Masse′E, Escorcia FE, Gottesman S. Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev, 2003,17(19):2374–2383.
[13] Antal M, Bordeau V, Douchin V, et al.A small bacterial RNA regulates a putative ABC transporter. J Biol Chem, 2005, 280(9):7901-7908.
[14] Ziolkowska K, Derreumaux P, Folichon M, et al.Hfq variant with altered RNA binding functions. Nucleic Acids Res, 2006, 34(2):709-720.
[15] Wilusz G, Wilusz J. Eukaryotic Lsm proteins: lessons from bacteria. Nat Struct Mol Biol, 2005 ,12(12):1031-1036.
[16] Butland G, Peregr?′n-Alvarez JM, Li J, et al.Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature, 2005 ,433(7025):531-537.
[17] Tsui HCT, Leung HCE, Winkler ME. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol, 1994, 13(1):35–49.
[18] Sonnleitner E, Hagens S, Rosenau F, et al.Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Micro Patho, 2003, 35(5):217-228.
[19] Nakao H, Watanabe H, Nakayama S, et al.Yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq). Mol Microbiol , 1995, 18(5):859–865.
[20] Robertson GT, Roop Jr RM. The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol, 1999, 34(4):690–700.
[21] McManus MT, Sharp PA. Gene Silencing in mammals by small interfering RNAs. Nature Reviews Genetics,2002, 3:737-747.
[22] Majdalani N, Vanderpool CK, Gottesman S. Bacterial Small RNA Regulators. Crit Rev Biochem Mol Biol, 2005,40:93–113.
[23] Gottesman S. Micros for microbes: Non-coding regulatory RNAs in bacteria. Trends Genet, 2005,21: 399–404.
[24] Storz G, Altuvia S, Wassarman KM. An abundance of RNA regulators. Annu Rev Biochem, 2005,74: 199–217.
[25] Pichon C, Felden B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci, 2005, 102: 14249–14254.
[26] Roberts C, Anderson KL, Murphy E, et al. Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on logphase mRNA half-lives. J Bacteriol, 2006, 188: 2593–2603.
[27] Novick RP, Ross HF, Projan SJ, et al. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J, 1993,12: 3967–3975.
[28] Cheung AL, Eberhardt KJ, Chung E, et al. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Invest, 1994, 94: 1815–1822.
[29] Gillaspy AF, Hickmon SG, Skinner RA, et al. Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun, 1995, 63: 3373–3380.
[30] Novick RP.Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[31] Morfeldt E, Taylor D, von Gabain A, et al. Activation of _-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J,1995,14:4569–4577.
[32] Huntzinger E, Boisset S, Saveanu C, et al. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J, 2005, 24: 824–835.
[33] Dunman PM, Murphy E, Haney S, et al.Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol, 2001, 183: 7341–7353.
[34] Novick RP.Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[35] Karzai A, Roche ED, Sauer RT. The SsrA–SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Boil, 2000,7: 449-455
[36] Karzai AW, Susskind MM, Sauer RT. SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA). EMBO J, 1999,18, 3793–3799.
[37] Vieira-da-Motta O, Ribeiro PD, Dias da Silva W, et al.RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production. Peptides, 2001, 22(10):1621-7.
[38] Balaban N, Goldkorn T, Gov Y, et al.Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein(TRAP). J Biol Chem, 2001, 276:2658– 26.
[39] Lindsey NS, Ing-Marie J, Vineet K, et al. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infection and Immunity, 2007,75(9): 4519-4527.
[40] Tsang LH, Daily ST, Weiss EC, et al. Mutation of traP in Staphylococcus aureus has no impact on expression of agr or biofilm formation. Infection and Immunity, 2007,75(9):4528-4533.
[41] Adhikari RP, Arvidson S, Novick RP. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infection and Immunity, 2007,75(9):4534-4540.
[42] Eric H, Sandrine B,Cosmin S. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression.The EMBO Journal ,2005, 24, 824–835.
[43] Sukhodolets MV, Garges S. Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq. Biochemistry, 2003,42: 8022–8034.
[44] Yang G, Gao Y, Dong J, et al.A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol Chem,2005,280:27431–27435.
[45] Singh VK, Jayaswal RK, Wilkinson BJ. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS MicrobiolLett.,2001,199:79-84.
[46] Wagner EGH, Altuvia S, Romby P .Antisense RNAs in bacteria and their genetic elements. Adv Genet ,2002,46: 361–398.
[47] Nicholson AW. Structure, reactivity, and biology of double-stranded RNA. Prog Nucleic Acid Res Mol Biol,1996,52:1–65.
[48] Dunn JJ. Ribonuclease III. In B. D. Boyer (ed.), The enzymes, Academic Press, New York, 1982,15:485-499.
[49] Fraser CM, Gocayne JD, White O, Adams MD .The minimal gene complement of Mycoplasma genitalium. Science,1995, 270(5235):397-403.
[50] Argaman L, Hershberg R, Vogel J,et al. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol,2001,11(12):941-50.
[51] Johannes HU, Jrg V. Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Research, 2007,doi:10.1093/nar/gkl1040.
[52] Bohn C, Rigoulay C, Bouloc P. No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiol, 2007,doi: 10.1186/1471-2180-7-10.
[53] Peng HL, Novick RP, Kreiswirth B, et al. Characterization, and Sequencing of an Accessory Gene Regulator (agr) in Staphylococcus aureus. J.Bacreriol, 1998, 170:4365-4372.
[54] Boisset S, Geissmann T, Huntzinger E, et al. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism. Genes Dev,2007, 21(11):1353-66.
[55] Schumacher MA, Pearson RF, M?ller T, et al. Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J. 2002 Jul 1;21(13):3546-56.
[2] Franze MT, Eoyang L, August JT. Factor fraction required for the synthesis of bacteriophage Qb-RNA.Nature,1968, 219(5154): 588-590.
[3] Moller T, Franch T, Hojrup P, et al.Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell , 2002, 9(1):23-30.
[4] Zhang A, Wassarman KM, Ortega J, et al.The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell ,2002,9(1):11-22.
[5] Sledjeski DD, Whitman C, Zhang A. Hfq is necessary for regulation by the untranslated RNA DsrA. J Bacteriol , 2001,183(6):1997-2005.
[6] Sun X, Zhulin I, Wartell R . Predicted structure and phyletic distribution of the RNA-binding protein Hfq. Nucleic Acids Res, 2002, 30(17):3662-3671.
[7] Schumacher MA, Pearson RF, Moller T, et al. Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J , 2002, 21(13):3546-3556.
[8] Mohanty BK, Maples VF, Kushner SR. The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Mol Microbiol, 2004, 54(4):905–920.
[9] Le Derout J, Folichon M, Briani F, et al.Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs. Nucleic Acids Res, 2003, 31(14):4017–4023.
[10] Hajnsdorf E, Re′gnier P. Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I. Proc Natl Acad Sci USA, 2000, 97(4):1501–1505.
[11] Vytvytska O, Jakobsen JS, Balcunate G, et al.Host factor I, Hfq binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability. Proc Natl Acad Sci USA, 1998, 95(24):14118–14123.
[12] Masse′E, Escorcia FE, Gottesman S. Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev, 2003,17(19):2374–2383.
[13] Antal M, Bordeau V, Douchin V, et al.A small bacterial RNA regulates a putative ABC transporter. J Biol Chem, 2005, 280(9):7901-7908.
[14] Ziolkowska K, Derreumaux P, Folichon M, et al.Hfq variant with altered RNA binding functions. Nucleic Acids Res, 2006, 34(2):709-720.
[15] Wilusz G, Wilusz J. Eukaryotic Lsm proteins: lessons from bacteria. Nat Struct Mol Biol, 2005 ,12(12):1031-1036.
[16] Butland G, Peregr?′n-Alvarez JM, Li J, et al.Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature, 2005 ,433(7025):531-537.
[17] Tsui HCT, Leung HCE, Winkler ME. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol, 1994, 13(1):35–49.
[18] Sonnleitner E, Hagens S, Rosenau F, et al.Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Micro Patho, 2003, 35(5):217-228.
[19] Nakao H, Watanabe H, Nakayama S, et al.Yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq). Mol Microbiol , 1995, 18(5):859–865.
[20] Robertson GT, Roop Jr RM. The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol Microbiol, 1999, 34(4):690–700.
[21] McManus MT, Sharp PA. Gene Silencing in mammals by small interfering RNAs. Nature Reviews Genetics,2002, 3:737-747.
[22] Majdalani N, Vanderpool CK, Gottesman S. Bacterial Small RNA Regulators. Crit Rev Biochem Mol Biol, 2005,40:93–113.
[23] Gottesman S. Micros for microbes: Non-coding regulatory RNAs in bacteria. Trends Genet, 2005,21: 399–404.
[24] Storz G, Altuvia S, Wassarman KM. An abundance of RNA regulators. Annu Rev Biochem, 2005,74: 199–217.
[25] Pichon C, Felden B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci, 2005, 102: 14249–14254.
[26] Roberts C, Anderson KL, Murphy E, et al. Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on logphase mRNA half-lives. J Bacteriol, 2006, 188: 2593–2603.
[27] Novick RP, Ross HF, Projan SJ, et al. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J, 1993,12: 3967–3975.
[28] Cheung AL, Eberhardt KJ, Chung E, et al. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Invest, 1994, 94: 1815–1822.
[29] Gillaspy AF, Hickmon SG, Skinner RA, et al. Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun, 1995, 63: 3373–3380.
[30] Novick RP.Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[31] Morfeldt E, Taylor D, von Gabain A, et al. Activation of _-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J,1995,14:4569–4577.
[32] Huntzinger E, Boisset S, Saveanu C, et al. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J, 2005, 24: 824–835.
[33] Dunman PM, Murphy E, Haney S, et al.Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol, 2001, 183: 7341–7353.
[34] Novick RP.Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[35] Karzai A, Roche ED, Sauer RT. The SsrA–SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Boil, 2000,7: 449-455
[36] Karzai AW, Susskind MM, Sauer RT. SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA). EMBO J, 1999,18, 3793–3799.
[37] Vieira-da-Motta O, Ribeiro PD, Dias da Silva W, et al.RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production. Peptides, 2001, 22(10):1621-7.
[38] Balaban N, Goldkorn T, Gov Y, et al.Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein(TRAP). J Biol Chem, 2001, 276:2658– 26.
[39] Lindsey NS, Ing-Marie J, Vineet K, et al. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infection and Immunity, 2007,75(9): 4519-4527.
[40] Tsang LH, Daily ST, Weiss EC, et al. Mutation of traP in Staphylococcus aureus has no impact on expression of agr or biofilm formation. Infection and Immunity, 2007,75(9):4528-4533.
[41] Adhikari RP, Arvidson S, Novick RP. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infection and Immunity, 2007,75(9):4534-4540.
[42] Eric H, Sandrine B,Cosmin S. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression.The EMBO Journal ,2005, 24, 824–835.
[43] Sukhodolets MV, Garges S. Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq. Biochemistry, 2003,42: 8022–8034.
[44] Yang G, Gao Y, Dong J, et al.A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol Chem,2005,280:27431–27435.
[45] Singh VK, Jayaswal RK, Wilkinson BJ. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS MicrobiolLett.,2001,199:79-84.
[46] Wagner EGH, Altuvia S, Romby P .Antisense RNAs in bacteria and their genetic elements. Adv Genet ,2002,46: 361–398.
[47] Nicholson AW. Structure, reactivity, and biology of double-stranded RNA. Prog Nucleic Acid Res Mol Biol,1996,52:1–65.
[48] Dunn JJ. Ribonuclease III. In B. D. Boyer (ed.), The enzymes, Academic Press, New York, 1982,15:485-499.
[49] Fraser CM, Gocayne JD, White O, Adams MD .The minimal gene complement of Mycoplasma genitalium. Science,1995, 270(5235):397-403.
[50] Argaman L, Hershberg R, Vogel J,et al. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol,2001,11(12):941-50.
[51] Johannes HU, Jrg V. Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Research, 2007,doi:10.1093/nar/gkl1040.
[52] Bohn C, Rigoulay C, Bouloc P. No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiol, 2007,doi: 10.1186/1471-2180-7-10.
[53] Peng HL, Novick RP, Kreiswirth B, et al. Characterization, and Sequencing of an Accessory Gene Regulator (agr) in Staphylococcus aureus. J.Bacreriol, 1998, 170:4365-4372.
[54] Boisset S, Geissmann T, Huntzinger E, et al. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism. Genes Dev,2007, 21(11):1353-66.
[55] Schumacher MA, Pearson RF, M?ller T, et al. Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J. 2002 Jul 1;21(13):3546-56.